Previous studies provided evidence that chronic ethanol ingestion leads to an increase in levels of the acidic phospholipids in synaptic membranes. Acidic phospholipids (PS, PA, PI, and poly-PI) are known to exert specific effects on membrane proteins due to their anionic properties. The polyphosphoinositides (PIP and PIP2) are of special interest due to their direct involvement in the signal transduction pathway. Using in vivo labeling protocols, our laboratory has obtained evidence that poly-PI turnover in rat and mouse brain was affected by acute and chronic ethanol administration. Furthermore, tolerance to the inhibitory effect of acute ethanol on the poly-PI pathway was observed in mice after chronic ethanol administration. Therefore, specific aim #1 is to further examine the intrinsic factors (e.g. duration and mode of ethanol administration) leading to development of tolerance in this signaling event. PI and PIP kinases are important enzymes in the signaling pathway since they provide the substrate (PIP2) for the receptor-mediated and G-protein coupled phospholipase C reaction. Preliminary studies with rat brain synaptic plasma membranes indicate that PIP kinase is more sensitive to ethanol in vitro than PI kinase. Therefore, specific aim #2 will include experiments to examine the mechanism underlying the action of ethanol on these two kinases and to test the hypothesis that chronic ethanol exposure results in an adaptive change in these kinases. Synaptic plasma membranes will be used to test for (a) effects of ethanol and aliphatic alcohols on the PI and PIP kinase activity in the presence and absence of exogenous substrates, (b) intrinsic changes in enzyme activity occurring in brain membranes after chronic ethanol administration, (c) interaction between the effects of ethanol, acidic phospholipids and cationic amphipathic compounds on purified PIP kinase, and (d) the effects of ethanol on the phosphomonoesterase activity. In order to relate ethanol's action on PI and PIP kinases to metabolism of the poly- PI cycle, specific aims #3 will use an astrocyte cell line as a model system. The effect of ethanol exposure on these enzymes will be related to the ability of these cells to respond to agonists (e.g. bradykinin) known to transduce signals through the poly-PI pathway. Information obtained from this project will be important in understanding the mechanism underlying the development of tolerance and may have the potential of targeting drugs or treatment protocols to alleviate the detrimental effects associated with alcohol withdrawal.